DU High throughput sequencing and rare diseases - Introduction to Ensembl Genome Browser/Biomart
Stephanie Le Gras\(^{1}\) - slegras@igbmc.fr
2023 January 25th
\(^1\) GenomEast platform, IGBMC
1 - Practical session: Ensembl Genome Browser
1.1 - Question
- What is the assembly version of the human genome available on the current Ensembl release?
Show answer
- How many coding genes does the Human genome contain in the current Ensembl release (v105)?
Show answer
- How many coding genes does the Human genome contain in the previous assembly (GRCh37 - Ensembl v75)?
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1.2 - Question
- Find out the size of the human chromosome 4 for assembly hg19 (GRCh37).
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1.3 - Question
Go back to http://ensembl.org.
- What is the version of the genome assembly of the Naked mole-rat (female)? (Heterocephalus glaber)
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- How many genes were predicted by Genscan?
Show answer
1.4 - Question
- How many alternative transcript gene BBS5 has (Human)
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- How many exons does transcript BBS5-205 have?
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- How many coding exons?
Show answer
Comment – Go back to the “Gene: BBS5” tab and have a look at the colors of transcripts BBS5-201 and BBS5-202 in the genome browser view
1.5 - Question
- Retrieve this page
Show answer
1.6 - Question
- Retrieve this page
Show answer
- Click on « configure this page » (left-bottom menu)
- Select Yes for « Show variations: »
1.7 - Question
Display RNAseq data for breast and skeletal muscle in the genome browser (in BRCA1 gene region).
Show answer
2 - Practical session: Biomart
2.1 - Retrieve information for 1 gene : IDH1
2.1.1 - Question
Using Ensembl/BioMart, retrieve all transcripts IDs and the gene ID of IDH1 gene (human). How many transcripts the gene IDH1 has? Use Ensembl Gene v105, for Human GRCh38.p13:
- Click on Filters :
- Expand the GENE section
- Select « Input external references ID list »
- Select HGNC symbol(s) in the drop down menu
- Enter IDH1 in the text box
- Click on Attributes :
- Select “Features” (top panel, selected by default)
- Select Gene stable ID, Transcript stable ID, Gene Name
Show answer
- Click on Filters (left panel)
- Expand the “GENE” section
- Select “Input external references ID list”, select “Gene Name(s)” in the drop down list and enter IDH1.
- Click on Count in the top left panel . You should get 1/67130 Genes.
- Click on Attributes (left menu)
- Select “Features” (selected by default)
- Select Gene stable ID, Transcript stable ID, Gene Name
- Click on Results (top left menu)
9 transcripts are found
2.1.2 - Question
Extract all exon sequences of the IDH1 gene in fasta format. Headers will contain the Gene names, Transcript stable IDs and Exon stable IDs.
Show answer
You can leave the Dataset and Filters the same, and go directly to the Attributes section:
- Click on Attributes (left panel)
- Select “Sequences”
- Expand the SEQUENCES section
- Select Exon sequences
- Expand “Header Information”
- Unselect “Gene stable ID” (Gene Information)
- Select Gene name (Gene Information), Transcript stable ID (Transcript Information) and Exon stable ID (Exon Information).
- Click on Results
2.1.3 - Question
Extract all coding sequences of the IDH1 gene in fasta format. Headers will contain the Transcript stable IDs and Exon stable IDs.
Show answer
You can leave the Dataset and Filters the same, and go directly to the Attributes section:
- Click on Attributes (left panel)
- In the SEQUENCES section: select Coding sequence
- In the “Header Information”: unselect Gene name (Gene Information) and keep Transcript stable ID (Transcript Information) and Exon stable IDs (Exon Information) selected.
- Click on Results
2.1.4 - Question
Retrieve GO-terms associated to the IDH1 gene (select GO Term Name, GO domain and GO Term Accession along with Gene stable ID, Transcript stable ID and Gene Name).
Show answer
You can leave the Dataset and Filters the same, and go directly to the Attributes section:
- Click on Attributes (left panel)
- Select “Features” (selected by default)
- In the GENE section: Gene stable ID, Transcript stable ID and Gene Name should be selected
- Expand the EXTERNAL section
- Select GO Term Name, GO domain and GO Term Accession
- Click on Results
2.1.5 - Question
Retrieve the germline variations found in this gene. Annotations to be found:
- Variant Name,
- Variant Alleles,
- Minor allele frequency,
- Chromosome/scaffold name,
- Chromosome/scaffold position start (bp),
- Chromosome/scaffold position end (bp),
- Variant Consequence along with Gene stable ID,
- Transcript stable ID
- Gene Name
Show answer
You can leave the Dataset and Filters the same, and go directly to the Attributes section:
- Click on Attributes (left panel)
- Select “Variant (Germline)”
- In the GENE section: Gene stable ID, Transcript stable ID and Gene Name should be selected
- Expand the GERMLINE VARIANT INFORMATION section
- Select:
- Variant Name,
- Variant Alleles,
- Minor allele frequency,
- Chromosome/scaffold name,
- Chromosome /scaffold position start (bp),
- Chromosome/scaffold position end (bp),
- Variant Consequence
- Click on Results
2.2 - Retrieve information for many genes
We have run an RNA-seq experiment and we have extracted upregulated genes. We are using RNAseq data from :
Strub, T., Giuliano, S., Ye, T., Bonet, C., Keime, C., Kobi, D., Le Gras, S., Cormont, M., Ballotti, R., and Bertolotto, C. (2011). Essential role of microphthalmia transcription factor for DNA replication, mitosis and genomic stability in melanoma. Oncogene 30, 2319–2332.
In this study, they compared the transcriptome in melanoma cell lines between cells with an siRNA against MITF or an siRNA against Luciferase (used as a control). The data are given as a TSV (Tab-Separated Values) file which contain the number of reads per genes. Genes are identified by their Ensembl gene IDs.
Data have been analyzed using the Human genome hg38/GRCh38 - Ensembl v95.
Here are the different column of the file to be analyzed:
- Gene id | Ensembl gene ID |
- siLuc2 | Raw read counts - control - 1st biological replicate |
- siLuc3 | Raw read counts - control - 2nd biological replicate
|
- siMitf3 | Raw read counts - siMITF - 1st biological replicate
|
- siMitf4 | Raw read counts - siMITF - 2nd biological replicate
|
- norm.siLuc2 | Normalized read counts - control - 1st biological
replicate |
- norm.siLuc3 | Normalized read counts - control - 2nd biological
replicate |
- norm.siMitf3 | Normalized read counts - siMITF - 1st biological
replicate |
- norm.siMitf4 | Normalized read counts - siMITF - 2nd biological
replicate |
- … | … |
2.2.1 - Question
Download and uncompress the file {{:training:simitfvssiluc-up.tsv.zip|siMitfvssiLuc.up.txt.zip}} to extract gene annotations using Ensembl/BioMart for those genes. Use the column Gene ID to extract annotations. Annotations to extract are:
- gene IDs
- chromosome
- start of gene
- end of gene
- strand
- Associated Gene Name
- gene type
Show answer
In Ensembl/BioMart, create a new request using an archive of Ensembl v95.
- Click on Filters (left panel)
- Expand the GENE section
- Select “Input external references ID list” and select “Ensembl Gene ID(s)” in the drop down list
- Click on “Browse” and select the file siMitfvssiLuc.up.txt
- Click on “Count” (top left button). You should have: 3663/64914 Genes
- Click on Attributes (left panel)
- Select “Features” (selected by default), select Ensembl Gene ID, Chromosome Name, Gene Start (bp), Gene End (bp), Strand, Associated Gene Name and Gene type.
- Click on Results
- Select Compressed file (.gz) in the drop down menu. Click on Go to download the resulting file.
2.2.2 - Question
You want to run a de novo motif discovery on all promoters of the upregulated genes (the ones from the file siMitfvssiLuc.up.txt). Extract the promoter sequences of all up-regulated genes: retrieve the 200nt upstream of the transcripts of these genes.
Show answer
Don’t change Dataset and Filters – simply click on Attributes.
- Click on Attributes (left panel)
- Select “Sequences”
- Expand the SEQUENCES section
- Select Flank (Transcript) and enter 200 in the Upstream flank text box
- Expand the Header information section
- Select, in addition to the default selected attributes, Description and Associated Gene Name
TIPS
Flank (Transcript) will give the flanks for all transcripts of a gene with multiple transcripts. Flank (Gene) will give the flanks for one possible transcript in a gene (the most 5’ coordinates for upstream flanking).2.3 - Retrieve information using genomic coordinates
Use Biomart of current version of Ensembl.
2.3.1 - Question
How many genes are located in the genomic region: 2:208226227-208276270.
Show answer
In Ensembl/BioMart, create a new request:
- Click on Filters (left panel)
- Expand the REGION section
- Select “Multiple Chromosomal Regions” and enter 2:208226227:208276270 in the text box
- Click on Count
2.3.2 - Question
Extract the coordinates of all human genes located on chromosomes (exclude scaffolds). Information to extract for each gene:
- Gene stable ID
- Chromosome/scaffold name
- Gene Start (bp)
- Gene End (bp)
- strand
- Gene Name
Show answer
In Ensembl/BioMart, create a new request (see slide 2.)
- Click on Filters (left panel)
- Expand the REGION section
- Select “Chromosome” and multiple select 1 -> MT (click and drag). This corresponds to 61487 / 68005 Genes
- Click on Attributes (left panel)
- Select “Features” (selected by default)
- In GENE, select Gene stable ID, Chromosome/scaffold name, Gene Start (bp), Gene End (bp), strand and Gene Name
- Click on Results
2.4 - Question
The following is a list of 11 IDs of human proteins from the NCBI RefSeq database:
- NP_001218
- NP_203125
- NP_203124
- NP_203126
- NP_001007233
- NP_150636
- NP_150635
- NP_001214
- NP_150637
- NP_150634
- NP_150649
Generate a list that shows to which Gene stable IDs and to which Gene names these RefSeq IDs correspond. Do these 11 proteins correspond to 11 genes?
Show answer
- Click New.
Choose the ENSEMBL Genes 105 database.
Choose the Homo sapiens genes (GRCh38.p13) dataset.
Click on Filters in the left panel.
Expand the GENE section by clicking on the + box.
Select ID list limit - RefSeq peptide ID(s) and enter the list of IDs in the text box (either comma separated or as a list).
HINT: You may have to scroll down the menu to see these.
Count shows 4 genes (remember one gene may have multiple splice variants coding for different proteins, that is the reason why these 11 do not correspond to 11 genes).
Click on Attributes in the left panel.
Select the Features attributes page.
Expand the External section by clicking on the + box.
Select Gene name and RefSeq Protein ID from the External References section.
Click the Results button on the toolbar.
Select View All rows as HTML or export all results to a file. Tick the box Unique results only.
2.5 - Bonus
Forrest et al performed a microarray analysis of peripheral blood mononuclear cell gene expression in benzene-exposed workers (Environ Health Perspect. 2005 June; 113(6): 801–807). The microarray used was the human Affymetrix U133A/B (also called U133 plus 2) GeneChip. The top 8 up-regulated probe-sets were:
- 207630_s_at
- 221840_at
- 219228_at
- 204924_at
- 227613_at
- 223454_at
- 228962_at
- 214696_at
2.5.1 - Question
Retrieve for the genes corresponding to these probe-sets the Gene and Transcript stable IDs as well as their Gene names and descriptions.
Show answer
- Click New.
Choose the ENSEMBL Genes 105 database.
Choose the Homo sapiens genes (GRCh38.p13) dataset.
Click on Filters in the left panel.
Expand the GENE section by clicking on the + box.
Select ID list limit - Affy hg u133 plus 2 probeset ID(s) and enter the list of probeset IDs in the text box (either comma separated or as a list).
Count shows 9 genes match this list of probesets.
Click on Attributes in the left panel.
Select the Features attributes page.
Expand the GENE section by clicking on the + box.
In addition to the default selected attributes, select Description.
Expand the External section by clicking on the + box.
Select Gene name from the External References section and AFFY HG U133-PLUS-2 from the Microarray Attributes section.
Click the Results button on the toolbar.
Select View All rows as HTML or export all results to a file. Tick the box Unique results only.
Your results should show that the 11 probes map to 9 Ensembl genes.
2.5.2 - Question
In order to be able to study these human genes in mouse, identify their mouse orthologues. Also retrieve the genomic coordinates of these orthologues.
Show answer
You can leave the Dataset and Filters the same, and go directly to the Attributes section:
Click on Attributes in the left panel.
Select the Homologs attributes page.
Expand the GENE section by clicking on the + box.
Select Associated Gene Name.
Deselect Ensembl Transcript ID.
Expand the ORTHOLOGS section by clicking on the + box.
Select Mouse Ensembl Gene stable ID, Mouse chromosome/scaffold name, Mouse chromosome/scaffold Start (bp) and Mouse chromosome/scaffold End (bp).
Click the Results button on the toolbar.
Check the box Unique results only. Select View All rows as HTML or export all results to a file.
Your results should show that for most of the human genes has least one mouse orthologue.
3 - Credits
Some exercices are taken from Ensembl tutorials.